This project is designed to further purify and characterize various forms of L-glutamate decarboxylase (GAD) which is the rate-limiting enzyme of gamma-aminobutyric acid (GABA), an important neurotransmitter, and to delineate the role of these enzymes in the regulation of effective level of GABA. Another important aspect of this project will be the production of antibodies against the purified GAD from various sources. This result will enable us to apply immunocytochemical methods to localize various forms of GAD at precise cellular and subcellular levels on tissue section, and also to add immunochemical methods to distinguish various forms of GAD, in addition to physical and chemical criteria. More specifically, I plan to perform the following studies: (1) Purify GAD (GAD I) from the enurons and GAD (GAD II) from the glia or non-neural tissues, e.g., kidney and heart by methods similar to those I had developed for the successful purification of GAD I from mouse brain and GAD II from calf heart (see Preliminary Results); (2) Perform immunochemical studies, e.g., immunodiffusion, inhibition of enzyme activities by antibodies, mincrocomplement fixation and immunoelectrophoresis to determine species-, tissue-, and cell-specificities of GAD I and II; (3) Localize GAD I and II on tissue section at light and electron microscopic levels by methods similar to those we had successfully developed for the visualization of GAD I in various regions of CNS in rat. When it becomes desirable, GAD I and II can be localized on the same tissue section by double staining technique; (4) Perform physical, chemical and enzymatic studies of GAD I and II and their subunits with special emphasis on the regulatory mechanism of GAD activities, the detailed structure of active site and the mechanism of decarboxylation; (5) Perform developmental studies of the various forms of GAD employing biochemical, immunochemical and morphological techniques. The preliminary results, which indicate that GAD II from calf heart has been highly purified and antibodies against this preparation are being produced, combined with the experience of the Principal Investigator, have shown the feasibility of the proposed project.